Consumable for laser capture microdissection

ABSTRACT

Systems and methods for acquiring laser capture microdissection samples are disclosed. An integral portion of a biological reaction vessel includes a transfer film carrier having a substrate surface; and a laser capture microdissection transfer film coupled to the substrate surface of the transfer film carrier. The systems and methods facilitate quick and accurate laser capture microdissection while simultaneously minimizing contamination.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part under 35 U.S.C. 120 ofcopending U.S. Ser. No. 60/060,732, filed Oct. 1, 1997, now pending, theentire contents of which are hereby incorporated by reference as iffully set forth herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention The present invention relates generallyto the field of laser capture microdissection (LCM). More particularly,the present invention relates to apparatus for acquiring LCM samplesthat include an LCM film mounted on at least a part of the interior ofan analysis container. Specifically, a preferred implementation of thepresent invention relates to a substantially planarized ethylene vinylacetate (EVA) polymer LCM film that is hot vacuum baked onto the bottomof a microcentrifuge tube cap. The present invention thus relates to anLCM sample acquisition apparatus of the type that can be termed planarcap.

[0003] 2. Discussion of the Related Art

[0004] Diseases such as cancer have long been identified by examiningtissue biopsies to identify unusual cells. The problem has been thatthere has been no satisfactory prior-art method to extract the cells ofinterest from the surrounding tissue. Currently, investigators mustattempt to manually extract, or microdissect, cells of interest eitherby attempting to mechanically isolate them with a manual tool or througha convoluted process of isolating and culturing the cells. Mostinvestigators consider both approaches to be tedious, timeconsuming, andinefficient.

[0005] A new technique has been developed which can extract a smallcluster of cells from a tissue sample in a matter of seconds. Thetechnique is called laser capture microdissection (LCM). Laser capturemicrodissection is a one-step technique which integrates a standardlaboratory microscope with a low-energy laser and a transparent ethylenevinyl acetate polymer thermoplastic film such as is used for the plasticseal in food product packaging.

[0006] In laser capture microdissection, the operator looks through amicroscope at a tissue biopsy section mounted on a standard glasshistopathology slide, which typically contains groups of different typesof cells. A thermoplastic film is placed over and in contact with thetissue biopsy section. Upon identifying a group of cells of interestwithin the tissue section, the operator centers them in a target area ofthe microscope field and then generates a pulse from a laser such as acarbon dioxide laser having an intensity of about 50 milliwatts (mW) anda pulse duration of between about 50 to about 500 milliseconds (mS). Thelaser pulse causes localized heating of the plastic film as it passesthrough it, imparting to it an adhesive property. The cells then stickto the localized adhesive area of the plastic tape directly above them,whereupon the cells are immediately extracted and ready for analysis.Because of the small diameter of the laser beam, extremely small cellclusters may be microdissected from a tissue section.

[0007] By taking only these target cells directly from the tissuesample, scientists can immediately analyze the gene and enzyme activityof the target cells using other research tools. Such procedures aspolymerase chain reaction amplification of DNA and RNA, and enzymerecovery from the tissue sample have been demonstrated. No limitationshave been reported in the ability to amplify DNA or RNA from tumor cellsextracted with laser capture microdissection.

[0008] Laser capture microdissection has successfully extracted cells inall tissues in which it has been tested. These include kidney glomeruli,in situ breast carcinoma, atypical ductal hyperplasia of the breast,prostatic interepithielial neoplasia, and lymphoid follicles. The directaccess to cells provided by laser capture microdissection will likelylead to a revolution in the understanding of the molecular basis ofcancer and other diseases, helping to lay the groundwork for earlier andmore precise disease detection.

[0009] Another likely role for the technique is in recording thepatterns of gene expression in various cell types, an emerging issue inmedical research. For instance, the National Cancer Institute's CancerGenome Anatomy Project (CGAP) is attempting to define the patterns ofgene expression in normal, precancerous, and malignant cells. Inprojects such as CGAP, laser capture microdissection is a valuable toolfor procuring pure cell samples from tissue samples.

[0010] The LCM technique is generally described in the recentlypublished article: Laser Capture Microdissection, Science, Volume 274,Number 5289, Issue 8, pp 998-1001, published in 1996, the entirecontents of which are incorporated herein by reference. The purpose ofthe LCM technique is to provide a simple method for the procurement ofselected human cells from a heterogeneous population contained on atypical histopathology biopsy slide.

[0011] A typical tissue biopsy sample consists of a 5 to 10 micron sliceof tissue that is placed on a glass microscope slide using techniqueswell known in the field of pathology. This tissue slice is a crosssection of the body organ that is being studied. The tissue consists ofa variety of different types of cells. Often a pathologist desires toremove only a small portion of the tissue for further analysis.

[0012] LCM employs a thermoplastic transfer film that is placed on topof the tissue sample. This film is manufactured containing organic dyesthat are chosen to selectively absorb in the near infrared region of thespectrum overlapping the emission region of common AlGaAs laser diodes.When the film is exposed to the focused laser beam the exposed region isheated by the laser and melts, adhering to the tissue in the region thatwas exposed. The film is then lifted from the tissue and the selectedportion of the tissue is removed with the film.

[0013] Thermoplastic transfer films such as a 100 micron thick ethylvinyl acetate (EVA) film available from Electroseal Corporation ofPompton Lakes, New Jersey (type E540) have been used in LCMapplications. The film is chosen to have a low melting point of about90° C.

[0014] The thermoplastic EVA films used in LCM techniques have beendoped with dyes, such as an infrared napthalocyanine dye, available fromAldrich Chemical Company (dye number 43296-2 or 39317-7). These dyeshave a strong absorption in the 800 nm region, a wavelength region thatoverlaps with laser emitters used to selectively melt the film. The dyeis mixed with the melted bulk plastic at an elevated temperature. Thedyed plastic is then manufactured into a film using standard filmmanufacturing techniques. The dye concentration in the plastic is about0.001 M.

[0015] While the films employed in LCM applications have provedsatisfactory for the task, they have several drawbacks. The opticalabsorption of a dye impregnated film is a function of its thickness.This property of the film may be in conflict with a desire to selectfilm thickness for other reasons.

[0016] The organic dyes which are used to alter the absorptioncharacteristics of the films may have detrimental photochemistry effectsin some cases. This could result in contamination of LCM samples. Inaddition, the organic dyes employed to date are sensitive to thewavelength of the incident laser light and thus the film must be matchedto the laser employed.

SUMMARY OF THE INVENTION

[0017] An object of the invention is to improve the speed of the lasercapture microdissection technique. Another object of the invention is toimprove the accuracy of the laser capture microdissection technique.Another object of the invention is to improve the reproducibility of thelaser capture microdissection technique. Yet another object of theinvention is to reduce the amount of contamination involved with thelaser capture microdissection technique. Therefore, there is aparticular need for an LCM consumable that integrates an LCM film intothe interior of an analysis container. A planar cap includes asubstantially planarized ethylene vinyl acetate (EVA) polymer LCM filmthat is hot vacuum baked onto the bottom of a microcentrifuge tube cap.The laser capture microdissection caps can be shipped as-baked (i.e.,packaged without post-bake processing) to protect the laser capturemicrodissection transfer film and minimize contamination. The cap, andthe configuration in which it is shipped, provides the additionaladvantages of quick and easy utilization. Thus, it is rendered possibleto simultaneously satisfy the requirements of speed, accuracy andresistance to contamination, which, in the case of the prior art, aremutually contradicting and cannot be simultaneously satisfied.

[0018] A first aspect of the invention includes a laser capturemicrodissection assembly comprising: a plate having a substantiallyplanar top surface; and at least one laser capture microdissection capconnected to said substantially planar top surface of said plate,wherein said at least one laser capture microdissection cap includes atransfer film carrier having a substrate surface; and a substantiallyplanarized laser capture microdissection transfer film connected to saidsubstrate surface of said transfer film carrier. A second aspect of theinvention includes a laser capture microdissection apparatus,comprising: a transfer film carrier having a substrate surface; and alaser capture microdissection transfer film coupled to said substratesurface of said transfer film carrier, said laser capturemicrodissection transfer film including at least one integrally formedstructural feature that protrudes and provides a controllable spacingbetween said laser capture microdissection transfer film and a sample. Athird aspect of the invention includes an integral portion of abiological reaction vessel, comprising: a transfer film carrier having asubstrate surface; and a laser capture microdissection transfer filmcoupled to said substrate surface of said transfer film carrier. Afourth aspect of the invention includes a laser capture microdissectionassembly comprising: a plate having a top surface; and at least onelaser capture microdissection cap coupled to said top surface of saidplate, wherein each of said at least one laser capture microdissectioncap includes a transfer film carrier having a substrate surface; and alaser capture microdissection transfer film coupled to said substratesurface of said transfer film carrier.

[0019] A fifth aspect of the invention includes a method of making thelaser capture microdissection assembly comprising: providing a platehaving a substantially planar top surface; providing at least one lasercapture microdissection cap, said at least one laser capturemicrodissection cap including a transfer film carrier having a substratesurface; providing a laser capture microdissection transfer filmadjacent to said substrate surface of said transfer film carrier; andhot vacuum baking said at least one laser capture microdissection capand said plate so as to substantially planarize said laser capturemicrodissection transfer film. A sixth aspect of the invention includesa method of making a laser capture microdissection consumable,comprising: providing a transfer film carrier having a substratesurface; and forming a laser capture microdissection transfer film onsaid substrate surface, wherein forming includes hot vacuum baking saidlaser capture microdissection transfer film. A seventh aspect of theinvention includes a method of making an integral portion of abiological reaction vessel, comprising: providing a transfer filmcarrier having a substrate surface; and fabricating a laser capturemicrodissection transfer film on said substrate surface. An eight aspectof the invention includes a method of making a laser capturemicrodissection assembly, comprising: providing a plate having a topsurface; providing at least one laser capture microdissection cap, saidat least one laser capture microdissection cap including a transfer filmcarrier having a substrate surface; providing, for said at least onelaser capture microdissection cap, a laser capture microdissectiontransfer film coupled to said substrate surface of said transfer filmcarrier; placing said at least one laser capture microdissection cap incontact with said plate; and hot vacuum baking both said at least onelaser capture microdissection cap and said plate so as to produce saidlaser capture microdissection assembly.

[0020] A ninth aspect of the invention includes a method of imaging asample with a microscope, comprising: providing said microscope;locating a scattering media within a beam path defined by saidmicroscope and within a few millimeters of a sample; and imaging saidsample through said scattering media with said microscope. A tenthaspect of the invention includes a microscope, comprising: a scatteringmedia located within a beam path defined by said microscope and within afew millimeters of a sample.

[0021] These, and other, aspects of the present invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingpreferred embodiments of the present invention and numerous specificdetails thereof, is given by way of illustration and not of limitation.Many changes and modifications may be made within the scope of thepresent invention without departing from the spirit thereof, and theinvention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] A clear conception of the advantages and features constitutingthe present invention, and of the components and operation of modelsystems provided with the present invention, will become more readilyapparent by referring to the exemplary, and therefore nonlimiting,embodiments illustrated in the drawings accompanying and forming a partof this specification, wherein like reference numerals (if they occur inmore than one view) designate the same elements. Consequently, theclaims are to be given the broadest interpretation that is consistentwith the specification and the drawings. It should be noted that thefeatures illustrated in the drawings are not necessarily drawn to scale.

[0023] FIGS. 1A-1C illustrate three views of a laser capturemicrodissection (LCM) sample plate, representing an embodiment of thepresent invention;

[0024] FIGS. 2A-2C illustrate three views of the sample plate shown inFIGS. 1A-1C after coating with a release agent, representing anembodiment of the present invention;

[0025] FIGS. 3A-3D illustrate four views of a sample carrier,representing an embodiment of the present invention;

[0026] FIGS. 4A-4D illustrate four views of the sample carrierillustrated in FIGS. 3A-3D after an LCM film is added, representing anembodiment of the present invention;

[0027] FIGS. 5A-5C illustrate three views of an assembly that includesfour of the sample carriers depicted in FIGS. 4A-4D and one of theplates depicted in FIGS. 2A-2C, representing an embodiment of thepresent invention;

[0028] FIGS. 6A-6C illustrate three views of a completed assembly aftervacuum hot cast molding, representing an embodiment of the presentinvention;

[0029] FIGS. 7A-7B illustrate two sequential views of a laser capturemicrodissection film with molded features, representing an embodiment ofthe present invention;

[0030]FIG. 8 illustrates a bottom view of a laser capturemicrodissection film with molded features, representing an embodiment ofthe present invention;

[0031]FIG. 9 illustrates a side view of a laser capture microdissectionapparatus, representing an embodiment of the invention;

[0032]FIG. 10 illustrates a side view of a microcentrifuge tube cap witha negative draft, representing an embodiment of the invention; and

[0033] FIGS. 11A-11D illustrates a several views of a biologicalreaction vessel, representing an embodiment of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0034] The present invention and the various features and advantageousdetails thereof are explained more fully with reference to thenonlimiting embodiments that are illustrated in the accompanyingdrawings and detailed in the following description. Descriptions of wellknown components and processing techniques are omitted so as not tounnecessarily obscure the present invention in detail.

[0035] The entire contents of U.S. Ser. No. ______, filed Feb. 7, 1997(Docket No. ARCT-002); U.S. Ser. No. 08/797,026, filed Feb. 7, 1997;U.S. Ser. No. 08/800,882, filed Feb. 14, 1997; U.S. Ser. No. 60/060,731,filed Oct. 1, 1997; and U.S. Ser. No. 60/060,732, filed Oct. 1, 1997 arehereby expressly incorporated by reference into the present applicationas if fully set forth herein.

[0036] Turning to FIGS. 1A-1C, a plate 100 is depicted. Plate 100 can befabricated from metal, glass, ceramic, or any other material suitablefor the subsequent processing steps described below. In a preferredembodiment, plate 100 is a glass microscope slide. It is important thatthe top surface 101 of plate 100 be flat. Although the depictedembodiment shows a bare microscope slide, the plate can be coated, orotherwise surface treated, in a preliminary processing step.

[0037] Turning now to FIGS. 2A-2C, the plate 100 is depicted with arelease agent 210. The release agent 210 is applied to the top surface101. It will be noted that the top surface 101 is obscured by therelease agent 210 in FIGS. 2A-2B but is clearly visible as an interfacein FIG. 2C.

[0038] The release agent can be any suitable nonadhesive material suchas, for example, silicones, or TEFLON (i.e., polytetrafluoroethylene).Advantageously, the release coating can be a surfactant that increasesthe contact angle of liquids with which it comes in contact. It isimportant that the release agent 210 maintain and extend the flatnessprovided initially by the top surface 101. In a preferred embodiment,the release agent 210 can include a silicone containing surfactant agentsuch as, for example, RAIN-X.

[0039] Turning now to FIGS. 3A-3D, a sample carrier 300 is depicted. Thesample carrier 300 has an upper portion 310 and a lower portion 320. Theupper portion 310 includes a top surface 315 and an outer perimeter 317,and a shoulder 319. The lower portion 320 includes a flare 322, an innerperimeter 324, a taper 326 and a substrate surface 328.

[0040] The sample carrier 300 can be a polymeric cap that is oftransparent optical quality. For example, the cap could be fabricatedfrom polycarbonate, or other suitable optically transparent plastic.However, the cap does not need to be optically transparent provided theabsorption characteristics of the polymer from which it is made arecompatible with suitable transmission of the laser energy to the capturefilm.

[0041] Turning now to FIGS. 4A-4D, a laser capture microdissection (LCM)transfer film 400 is shown being applied to the sample carrier 300. Itwill be appreciated that the LCM transfer film 400 is depicted out ofscale for the sake of clarity. The laser capture microdissectiontransfer film 400 can be applied to the bottom of a circular cap bypunching a circular section from a free-standing sheet of ethylene vinylacetate. Alternatively, the LCM transfer film 400 can be molded to thebottom of the cap. The LCM transfer film 400 can be deposited on the capusing a process such as spin coating, dipping, or spraying. In anyevent, manufacture of the consumable should be carried out in a sterileenvironment.

[0042] It is advantageous that the LCM transfer film 400 be thin. Forexample, a 50 micron thick film is preferable to a 100 micron thickfilm. However, the film can advantageously be fabricated in thicknessesof approximately 500, 400, 300, 200, 100, 50 microns, or less.

[0043] Turning to FIGS. 5A-5C, a plurality of combined sample carriers300 together with their corresponding LCM transfer films 400 aredepicted being lowered toward the release agent 210 that is coated ontop of the plate 100. The LCM transfer films 400 can be an ethylenevinyl acetate (EVA) polymeric material. It will appreciated that FIG. 5Adepicts the assembly process at an earlier point in time compared toFIG. 5C wherein the gap between the LCM transfer film 400 and therelease agent 210 is almost closed.

[0044] Turning now to FIGS. 6A-6C, the assembly of four sample carriers300 on plate 100 is depicted during the process step of vacuum hotbaking. The process of vacuum hot baking causes the EVA to soften, meltand flow thereby conforming to the substantially planar surfacepresented by the release agent 210. In this way, the flatness possessedby the plate 100 is transferred to the LCM transfer film 400. This alsoeliminates trapped air.

[0045] The hot vacuum baking of the film can take place in moderatevacuum. In a preferred embodiment, the hot cast molding takes place atone torr and 95 degrees C for approximately one hour.

[0046] In an alternative embodiment, instead of attaching the LCM filmto the base of the cap prior to its placement on top of the releaseagent coated plate, the LCM film can be coated on top of the releaseagent as a film layer. A sample carrier can then be placed on top of theLCM film. An assembly of one, or more, such combinations can then besubjected to hot vacuum melt casting to planarize at least that portionof the LCM film that is located at the interface between the samplecarrier and the release agent. In this way, when the sample carrier isremoved from the plate, a portion of the planarized LCM film thatcorresponds with the bottom surface of the sample carrier will be brokenaway from the assembly together with the cap that is being removed.Those portions of the LCM film that are not adjacent the bottom of thecap being removed will remain on the plate. In a preferred embodiment,when the sample carrier is pulled away from the plate, a twisting motionis applied to the sample carrier either before and/or during linearseparation of the two prime components so as to exert a sheer force bothwithin the LCM film and between the LCM film and the release layer.

[0047] The release coating can be a silicone. Alternatively, the releasecoating can be a polytetrafluoroethylene.

[0048] Throughout this specification, the more descriptive phrase“transfer film carrier” can be substituted for the phrase “samplecarrier.” In general, the transfer film carrier carries the transferfilm. Only that portion of the sample that is transferred to thetransfer film is carried by the carrier.

[0049] The ethylene vinyl acetate can be selected from among theavailable materials based on the following criteria. The ethylene vinylacetate should have a high melt index. A high melt index is indicated bylow viscosity and low molecular weight.

[0050] It is important that the ethylene vinyl acetate, or othermaterial being used for the LCM transfer film, have a modest tack. Thus,the transfer film is somewhat sticky but will not bind to everythingwith which it comes in contact.

[0051] The caps can be made from clear plexiglass G (i.e., polymethylmethacrylate). By treating the glass slide with a surfactant before thecaps are vacuum hot cast in place, the completed caps can be popped offthe glass slide just before they are needed for acquisition of samplematerial.

[0052] In a preferred embodiment, the cap is sized to fit in a standardmicrocentrifuge tube. The LCM transfer film can be attached to the capusing glue, or by welding the thermoplastic, or by some other mechanicalmeans, holding the film in place.

[0053] The side walls of the cap can have a negative draft. Thisnegative draft can be machined into the tooling with which the caps aremade.

[0054] After capturing the tissue to be analyzed on the bottom of thecap, the cap is placed on the microcentrifuge tube containing proteinase(i.e., protease, e.g., Trypsin) solution and the tube is inverted. Thetissue is then dissolved and the DNA is free to enter the solution. Thesolution is then pipetted out of the tube and into the PCR mixture.

[0055] While not being bound by theory, it is believed that the EVA filmexpands both up and down when it is exposed to the energy from thelaser. As an approximation, it is believed that the EVA film expandsapproximately 12-15% downward and upward when it is exposed to the LCMcharge from the laser. The upward expansion is restricted by the plasticcap.

[0056] The thickness of the LCM transfer film should be held to within20%, preferably 5%. The bottom, exposed surface of the LCM transfer filmcan be termed a capture surface. The flatness of the LCM transfer filmshould be held to within approximately five microns, preferablyapproximately one micron. The flatness of the film can readilycharacterized based on the number of fringes multiplied by λ/2. Theflatness of the LCM transfer film should preferably be held to withintwo waves which is approximately equal to ¼ micron per fringe, given a λof 540 nm.

[0057] The dye in the ethylene vinyl acetate is what absorbs the laserenergy. The ethylene vinyl acetate transforms to a liquid phase, infusesinto the cell structure of interest and then hardens.

[0058] The particular manufacturing process used for fabricating theassembly should be inexpensive and reproducible. Conveniently, thefabrication of the present invention can be carried out by using anycoating and baking method. It is preferred that the process be conductedin a contaminant-free environment. For the manufacturing operation, itis moreover an advantage to employ an automated method.

[0059] However, the particular manufacturing process used forfabricating the assembly is not essential to the present invention aslong as it provides the described assembly. Normally those who make oruse the invention will select the manufacturing process based upontooling and energy requirements, the expected application requirementsof the final product, and the demands of the overall manufacturingprocess.

[0060] The particular material used for the cap should be biologicallyand chemically inert. Conveniently, the cap of the present invention canbe made of any material with a melting point higher than that of EVA. Itis preferred that the material be inexpensive. For the manufacturingoperation, it is moreover an advantage to employ a transparentthermoplastic material that can be injection molded or machined. Forexample, the cap can include polymethyl methacrylate. By properselection of the polymeric materials, the cap can be solid. There is noneed for a through-hole through the center axis of the cap.

[0061] However, the particular material selected for the cap is notessential to the present invention, as long as it provides the describedfunction. Normally, those who make or use the invention will select thebest commercially available material based upon the economics of costand availability, the expected application requirements of the finalproduct, and the demands of the overall manufacturing process.

[0062] The LCM transfer film can be any suitable thermoplastic. Forexample, the LCM transfer film can include one or more of: EVAs;polyurethanes (PU); polyvinyl acetates; ethylene-methyl acrylate (EMAC);polycarbonate (PC); ethylene-vinyl alcohol copolymers (EVOH);polypropylene (PP); and expandable or general purpose polystyrene (PS).ELVAX 410, 200 and 205 are suitable resins of EVA that are commerciallyavailable from DuPont wherein the operative variant is the amount ofvinyl.

[0063] The LCM transfer film can include an absorptive substance. Theabsorptive substance can include an absorptive dye. This dye can beeither a broad band absorptive dye or a frequency specific absorptivedye. For example, the absorptive dyes can include one or more of:tin(IV) 2,3-naphthalocyanine dichloride; silicon(IV)2,3-naphthalocyanine dihydroxide; silicon (IV) 2,3-naphthalocyaninedioctyloxide; and vanadyl2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine. Also, the absorptivesubstance can include a plurality of Fullerines (i.e., Bucky Balls,e.g., C60).

[0064] The LCM transfer film can also include a scattering media. Sincethe LCM transfer film is very close to the sample, the scattering mediareduces shadows, thereby improving the process of imaging. Thescattering media can include a diffusing material. For example, the LCMtransfer film can be loaded with a small particulate material thatscatters the illumination light so as to minimize shadows and improveimaging without detrimentally effecting the LCM beam. Alternatively, thetransfer film can include a dichromatic gelatin (DCG) to perform thesame functions. The DCG can be exposed and developed to provide specificdiffuser properties within the transfer film such as shaping.

[0065] There are a variety of techniques for building a noncontact LCMtransfer film and/or carrier. The purpose of the noncontact LCM approachis to provide a method for the elimination of problems associated withnonspecific binding of tissue to an LCM film. In more detail, if asample slide has areas with loosely attached cells, these portions ofthe sample can be lifted mistakenly from the slide due to nonspecificattachment to the LCM film. That is, these areas stick to the film eventhough they were not illuminated by the laser. If these portions aretransferred to the reagent vessel they will be digested by the reagentsand appear as contaminants in the sample. It is important to prevent theloosely bound tissue areas from contacting the film.

[0066] One method for preventing the contact of the film to areas oftissue that might nonspecifically transfer is to offset (distance) thefilm a few microns from the tissue sample. In the area illuminated bythe laser, the film expands roughly 10% of its thickness (about 5 to 10microns based on a typical thickness of 50 to 100 microns) and contactsthe tissue, thereby allowing transfer in the illuminated region. Outsidethis region, the film and tissue never come in contact because the filmis spaced away from the tissue. The film, however, must not be spacedtoo far from the tissue (greater than a few microns) since the filmneeds to contact the tissue after its expands due to the laserillumination.

[0067] One technique to make a noncontact LCM transfer film that“stands-off” a few microns is to create a series of pedestals that are afew microns high so as to provide a series of standoffs for the cap torest on. These pedestals can be created by exposing edges of thetransfer film to the focused laser beam. The laser beam distorts thenormally flat film in the focal region raising the surface in thisregion. By placing these pedestals at the vertices of an equilateraltriangle with points located at the rim of the transfer film carrier agood three-point mount is provided. The height of these pedestals can beadjusted by changing the power and pulse length of the focused laserbeam. The diameter can be adjusted by changing the diameter of the laserbeam. The exposure levels are similar to the levels used for tissuetransfer: approximately 10-90 mW for approximately 10-90 milliseconds.(To create the pedestals it may help to expose the film when it is incontact with a glass slide.) The reagent vial can be constructed so thatit has an internal rim that contacts the pedestals, sealing them fromthe reagent, thereby preventing tissue that might be on the pedestalsfrom contaminating the sample.

[0068] Turning now to FIGS. 7A-7B, an LCM film 700 can be provided withfeatures 710. The features 710 can include a raised portion 720(pedestal) and a protruding feature 730 (e.g., rim). The features 710can be molded (e.g., replicated), or otherwise formed (e.g., by laser),in the LCM film 700. Such features give the LCM film 700 a workingsurface that defines a topography.

[0069] The purpose of the features 710 is to provide an additional wayof selecting single cells from a tissue sample using LCM, other thanjust a very small laser spot size. The features 710 that are fabricatedinto the LCM transfer film can be roughly the size of a desired cell740. The features 710 can extend out from the film surface for adistance of several microns.

[0070] The film 700 itself can be offset from the cells a distance offrom approximately 5 to approximately 10 microns by the protrudingfeature 730 that runs around the circumference of the cap. To stabilizethe plane of the film, it will be appreciated that the protrudingfeature only needs to extend along at least three points of a perimeterof the film and does not need to be a continuous rim.

[0071] The features 710 can be fabricated by hot cast molding the LCMfilm 700 against a mold that has complimentary shapes of the featureslaser machined into the mold surface. Such a mold can be made out of apolished metal surface or a glass surface using a Q-switched laserfocused to a diameter of from approximately 5 to approximately 20microns. The features 710 can also be fabricated by molding the filmagainst a mold surface that is micromachined with a diamond stylus. Thetopography is transferred from the mold to the film via replication.

[0072] A protuberance (raised portion 720) for acquiring the desiredcell 740 can include a small raised area of LCM film roughly 5 to 20microns in diameter. When a laser beam 750 heats this portion of thefilm, the raised portion 720 will contact the tissue first and the laserpower can be adjusted so that the surrounding adjacent film regions donot contact the tissue. Thus, the raised portion 720 provides spatialdiscrimination in addition to the spatial discrimination provided by theposition, size and mode of the laser beam. An advantage of the features710 is that a larger laser beam could be used and a researcher orlaboratory technician could still achieve single cell lift-off. Theraised portion of the film (raised portion 720) will be heated to ahigher temperature than the surrounding flat film area. The protrudingfeature 730 (i.e., the rim) will not be heated. This would also increasethe likelihood that a cell in the region of the feature would becaptured exclusively. Of course, it is advantageous that raised portion720 not protrude as far as protruding feature 730.

[0073] Referring now to FIG. 8, multiple pedestals 800 could be moldedinto an LCM film 810 to allow multiple single cell lift off regions. TheLCM film 810 could again include a rim 820. Multiple cells could then beanalyzed in a single microcentrifuge tube.

[0074] The structural feature (i.e., spacer) that holds the film awayfrom the sample can be hot vacuum baked into the transfer film.According to this process, a negative of the structural feature can beformed in a plate. The structural feature is then replicated (as apositive) in the film when it is heated and flows into the void definedby the negative of the feature. Alternatively, the structural featurecan be formed in the transfer film with the use of a laser, or even withmicro-machining equipment.

[0075] The structural feature, or spacer, can be integrally formed inthe laser capture microdissection transfer film. The structural featureprovides a separation between the transfer film and the sample. Thisseparation holds the film away from the sample, thereby enablingnoncontact laser capture microdissection.

[0076] The transfer film can be connected to the substrate surface witha refractive index matching transparent fluid or glue. Alternatively,the transfer film can be coupled to the substrate surface by punchingboth the sample carrier and the transfer film from stock materialsimultaneously. It is even possible to couple the film to the carrierwith double-sided tape.

[0077] The laser capture microdissection transfer film includes asubstantially planarized low land area. This low land area can beprovided with structural features that protrude so as to define a lasercapture microdissection acquisition zone. These protrusions can betermed pedestals. The low land can also be provided with structuralfeatures that hold most of the film away from the sample. In order tosupport the plane of the film, it is preferable to have at least threesuch supporting features. If these supporting features run around most,or all, of a perimeter of a transfer film, they can be termed a rim.

[0078] Whatever contacts the tissue needs to be equidistant from thetissue so that the dosimetry is constant across the transfer film. Inthis way, a known distance between the tissue and the transfer film canbe established. In many cases such a known distance will be fixed acrosssubstantial portions of the transfer film surface. However, it issufficient that the distance be known and does not need to be fixed. Thedistance needs to be known for the purpose of adjusting laser power soas to achieve tissue transfer.

[0079] When the transfer film is exposed to the electromagnetic energy,it expands (both up and down) against the substrate surface and contactsthe tissue, thereby injecting itself into the sample. In the case wherethere is a space between the transfer film and the top surface of thesample, (noncontact laser capture microdissection) the expanding filmwill be projected through that space before it contacts the top surfaceof the sample at the beginning of the injection phase.

[0080] Referring now to FIG. 9, a scatter illuminator design for an LCMdevice is illustrated. The purpose of the scatter illuminator design isto provide a more appropriate illuminator for an LCM microscope thatgenerates a more even illumination to prevent shadows from obscuringinternal cell structure.

[0081] A laser capture microdissection apparatus includes a top portion910 and a bottom portion 920. The top portion 910 includes an uppersurface to which a scattering media 930 can be coupled. The bottomportion 920 includes a substrate surface to which a scattering media 940can be coupled. Either, or both, of the scattering media 930 and 940 canbe used. The scattering media can be incorporated into the transfer filmcarrier and/or the LCM transfer film.

[0082] Using a standard inverted microscope light source and placing ascattering media (e.g., a piece of paper) near the tissue to scatter thelight results in dramatically improved illumination of the sample andmuch better visualization. A scattering media of this type eliminatesthe need for refractive index matching of the sample. Such a scatteringmedia can allow visualization of the cell nucleus and other subcellularstructures that would normally be obscured by normal illuminationtechniques.

[0083] The scattering media can be a diffuser material. A diffusermaterial that is suitable for use as the scattering media is milk glasswhich is a very dense, fine diffuser available from Edmund Scientific asPart No. P43,717. Standard laser printer/photocopier paper can even beused as the scattering media. Other types of transparent scatteringmedia can be used, such as, for example, frosted glass, a lenticularsheet, a volume diffuser, and/or a surface diffuser. In any event, thescattering media should be a material that aggressively scatters theillumination light. A single sheet of typical ground glass is generallyinadequate and needs to be combined in multiple layers as a serial stackof three or four sheets of ground glass to diffuse the illuminationlight sufficiently.

[0084] The scattering media can be directly or indirectly connected tothe transfer film carrier and/or the LCM transfer film. Alternatively,the scattering media can be formed on a surface of, or the interior of,the transfer film carrier and/or the LCM transfer film. The scatteringmedia can be fabricated so as to shape the LCM beam and/or theillumination beam. The scattering media needs to be within a fewmillimeters of the sample to be effective. A few millimeters means lessthan one centimeter, preferably less than five millimeters.

[0085] Referring now to FIG. 10, a laser capture microdissectionapparatus 1000 is illustrated. The apparatus 1000 includes a top portion1010 and a bottom portion 1020. The bottom portion 1020 includes anegative draft 1030. The negative draft 1030 is preferably approximately5°. The bottom portion 1020 also includes a chamfer 1040. The chamfer1040 is preferably approximately 20°. The bottom portion 1020 alsoincludes a girdle 1050. The width of the girdle 1050 for line contactwith the interior of an analysis vessel is preferably approximately0.01″. Caps with a negative draft can be fabricated with a break-apartplastic injection molding die. Alternatively, negative draft caps can befabricated by interpolation with computer numeric control cutting toolmachinery.

[0086] Turning now to FIGS. 11A-11D, a laser capture microdissection(LCM) biological reaction vessel 1100 including an analysis vessel 1110with an internal ridge and a cap 1120 with a transfer film 1130. Thetransfer film 1130 can include EVA and can have a stand-off rim 1150.Stand-off rim 1150 can be a 10-20 micron ridge providing a noncontactregion in the center of the transfer film 1130. The cap 1120 is anintegral portion of the biological reaction vessel 1100. The analysisvessel 1110 is formed to include an internal ridge 1140. The internalridge slopes back toward an opening in the analysis vessel 1110 so as tomake a tight seal with the cap 1120, even if the stand-off rim is notpresent. The purpose of combining the internal ridge 1140 with thestand-off rim 1150 in a single embodiment is to provide an LCM analysisvessel and film carrier that have features to facilitate a noncontactmethod for positioning the transfer film over the tissue sample. The LCMnon-contact method reduces the probability that areas of tissue outsidethe focal adhesion region will be transferred. However, if the stand-offrim 1150 later comes in contact with the reaction, this advantage willbe lost. The analysis vessel 1110 with this internal sealing featureallows the transfer film 1130, with stand-off rim 1150, to contact thetissue but not contact reaction fluid in the analysis vessel 1110.

[0087] The biological reaction vessel 1100 includes the cap 1120 (lid)that can be removably coupled to the analysis vessel 1110. The transferfilm 1130 is attached to the clear plastic cap 1120. The transfer film1130 can be hot cast molded to include the stand-off rim 1150 that is 10microns thicker than the central region of the cap 1120. The stand-offrim 1150 can be termed an annular rim. The transfer film 1130 expands inthe region of the focused laser beam and is able to bridge the 10 microngap, thereby contacting the tissue and allowing transfer of a portion ofthe tissue to the film. This stand-off rim 1150 can be termed a standoffregion and acts as a spacer elevating the central region of the transferfilm 1130 above the tissue and preventing the transfer film 1130 fromcontacting the tissue in this central region, until the LCM laseractivate the transfer film 1130. This stand-off region feature can bemolded into the transfer film 1130 by pressing the transfer film 1130onto a heated plate that contains an inverse image of this step (spacer)feature. This method replicates the feature. Such a mold could beconstructed using a polished metal plate and standard chemical etchingtechniques. It could also be manufactured using glass or siliconsubstrates and chemical etching. Alternatively, a diamond lathe could beused to machine this feature onto a suitable metal substrate (e.g.,copper, aluminum, steel, etc.).

[0088] The cap 1120 that seals the liquid reagent analysis vessel 1110can be made out of inert plastic such as polypropylene or polyethylene.The analysis vessel 1110 has the internal ridge 1140 (step) that isdesigned to mate with and cover the annular rim of the cap 1120providing a tight seal at this point. This seal prevents liquids in theanalysis vessel 1110 from contacting the bottom surface of the rim ofthe cap. This design eliminates nonspecific tissue transfer since thestand-off rim 1150 is the only area of the cap 1120 that contacts thetissue (other than the desired transfer regions illuminated by thelaser) and the digestion reagents in the analysis vessel 1110 nevercontact this region (stand-off rim 1150). The internal ridge 1140feature in the analysis vessel can be designed with a slight angle so asto partially cut into the Transfer film 1130 providing a very tight sealsimilar to vacuum flange sealing techniques. A slight bulge orindentation can be molded into the barrel of the cap 1120 or into thetop portion of the analysis vessel 1110 so as to provide a downwarddirected force and a positive seal between the cap 1120 and the analysisvessel 1110.

EXAMPLE

[0089] A specific embodiment of the present invention will now befurther described by the following, nonlimiting example which will serveto illustrate in some detail various features of significance. Theexample is intended merely to facilitate an understanding of ways inwhich the present invention may be practiced and to further enable thoseof skill in the art to practice the present invention. Accordingly, theexample should not be construed as limiting the scope of the presentinvention.

[0090] In an exemplary embodiment of the invention, a glass microscopeslide is first cleaned. Then the glass microscope slide is spray coatedwith a thin layer of a commercially available silicone release agent, inthis example a silicone containing surfactant that is readilycommercially available (i.e., RAINEX). Meanwhile, a supply of samplecarriers in the form of microcentrifuge tube caps are molded fromplexiglass G. Cylindrical chips of LCM film punched from a sheet ofethylene vinyl acetate (EVA) are then attached to the bottom surface ofthe caps, optionally with an epoxy adhesive. The resultant capsubassemblies are then placed on top of the release agent coated glasssubassembly for hot vacuum baking. The hot vacuum baking is carried outat a pressure of approximately one torr or less at a temperature of 95°C. for approximately one hour. This planarizes the transfer film. Thebaked assembly is then allowed to cool to room temperature. Theresulting assembly can include a planoconcave void located between eachof the caps and the underlying plate. In this way only the perimeter ofthe bottom of the caps is in contact with the glass plate. This providestwo significant advantages. First, the working surface of the LCM filmis spaced apart from the glass slide in a vacuum and remains free ofsurface damage and contaminants. Second, the removal of each cap fromthe glass slide is facilitated by the fact that only a fraction of thesurface area of the bottom of the cap is attached to the release layerthat has been coated on the glass slide. Therefore, removal of the capfrom the slide requires much less force than if the entire lower surfaceof the cap were in contact with the release layer.

[0091] It can be appreciated that by both making and shipping the cap onthe same glass slide, the number of processing and packaging steps isreduced while reproducibility and cleanliness are improved.

[0092] The completed consumable products can be sterilized (e.g., withbeta or gamma radiation). Finally, the completed consumable productsshould be subjected to a rigorous quality assurance inspection.

[0093] There are a number of advantages to leaving the caps on the slideuntil they are about to be used. These advantages include protection ofthe optically flat surface. For example, leaving the caps on the slidereduces hydroxyl contamination of the transfer film. These advantagesalso include the prevention of particulate matter from settling on thesurface.

Practical Applications of the Invention

[0094] A practical application of the present invention that has valuewithin the technological arts is the collection of a large database ofgene expression patterns of both healthy and diseased tissue, atdifferent stages of diseases. This database will be used to more fullyunderstand that pathogenesis of cancer and infectious diseases. Thepresent invention will enable a scientist to identify gene patterns andincorporate this information into effective diagnostics for disease. Thepresent invention will allow medical doctors to compare actual patienttissue samples with archived data from patient samples at differentdisease stages, thereby allowing them to prescribe more effective stagetherapies, eliminate unnecessary procedures, and reduce patientsuffering. Other research areas where the present invention will finduse are drug discovery, developmental biology, forensics, botany, andthe study of infectious diseases such a drug-resistant tuberculosis.There are virtually innumerable uses for the present invention, all ofwhich need not be detailed here.

Advantages of the Invention

[0095] Laser capture microdissection, representing an embodiment of theinvention can be cost effective and advantageous for at least thefollowing reasons. The present invention will replace current methodswith better technology that allows for more accurate and reproducibleresults. The present invention can be used to provide a low costinjection molded polymer disposable that integrates a laser capturemicrodissection film into the interior surface of an analysis containersuch as a microcentrifuge tube.

[0096] All the disclosed embodiments of the invention described hereincan be realized and practiced without undue experimentation. Althoughthe best mode of carrying out the invention contemplated by theinventors is disclosed above, practice of the present invention is notlimited thereto. It will be manifest that various additions,modifications and rearrangements of the features of the presentinvention may be made without deviating from the spirit and scope of theunderlying inventive concept. Accordingly, it will be appreciated bythose skilled in the art that the invention may be practiced otherwisethan as specifically described herein.

[0097] For example, the individual components need not be formed in thedisclosed shapes, or assembled in the disclosed configuration, but couldbe provided in virtually any shape, and assembled in virtually anyconfiguration. Further, the individual components need not be fabricatedfrom the disclosed materials, but could be fabricated from virtually anysuitable materials. Further, although the caps and cap assembliesdisclosed herein are described as a physically separate module, it willbe manifest that the caps and cap assemblies may be integrated intoother apparatus with which they are associated. Furthermore, all thedisclosed elements and features of each disclosed embodiment can becombined with, or substituted for, the disclosed elements and featuresof every other disclosed embodiment except where such elements orfeatures are mutually exclusive.

[0098] It is intended that the appended claims cover all such additions,modifications and rearrangements. The claims are not to be construed asincluding means-plus-function limitations, unless such limitations areexplicitly recited using the term “means” in the claims. Expedientembodiments of the present invention are differentiated by the appendedsubclaims.

What is claimed is:
 1. A laser capture microdissection apparatus,comprising: a transfer film carrier having a substrate surface; and alaser capture microdissection transfer film coupled to said substratesurface of said transfer film carrier, said laser capturemicrodissection transfer film including at least one integrally formedstructural feature that protrudes and provides a controllable spacingbetween said laser capture microdissection transfer film and a sample.2. The laser capture microdissection apparatus of claim 1 , wherein saidlaser capture microdissection transfer film includes a material, thatupon exposure to sufficient electromagnetic energy, expands and projectsitself away from said substrate surface.
 3. The laser capturemicrodissection apparatus of claim 1 , further comprising a scatteringmedia in proximity to said laser capture microdissection transfer film.4. The laser capture microdissection apparatus of claim 1 , wherein saidlaser capture microdissection transfer film includes an absorptivesubstance.
 5. The laser capture microdissection apparatus of claim 1 ,wherein said laser capture microdissection transfer film is hot vacuumbaked onto said substrate surface.
 6. The laser capture microdissectionapparatus of claim 1 , wherein said laser capture microdissectiontransfer film is bonded to said substrate surface with a refractiveindex matching transparent glue.
 7. The laser capture microdissectionapparatus of claim 1 , wherein said transfer film carrier includes anegative draft such that a distal diameter defined by said surface ofsaid transfer film carrier is greater than a proximal diameter definedby an inner perimeter of said transfer film carrier.
 8. The lasercapture microdissection apparatus of claim 7 , wherein said transferfilm carrier includes a girdle that is contiguous with said negativedraft.
 9. The laser capture microdissection apparatus of claim 7 ,wherein said transfer film carrier includes a chamfer that is contiguouswith said substrate surface.
 10. The laser capture microdissectionapparatus of claim 1 , wherein said laser capture microdissectiontransfer film has a thickness that is less than 500 microns.
 11. Thelaser capture microdissection apparatus of claim 1 , wherein said lasercapture microdissection transfer film has a thickness that is held towithin 20%.
 12. The laser capture microdissection apparatus of claim 1 ,wherein said laser capture microdissection transfer film has a capturesurface that is opposite said substrate surface, said capture surfacehaving a flatness that is held within five microns.
 13. The lasercapture microdissection apparatus of claim 1 , wherein said lasercapture microdissection transfer film includes at least one pedestalthat protrudes and defines a laser capture microdissection acquisitionzone.
 14. The laser capture microdissection apparatus of claim 1 ,wherein said laser capture microdissection transfer film includes aprotruding feature that runs along at least three points of a perimeterof said laser capture microdissection transfer film.
 15. Amicrocentrifuge tube cap comprising the laser capture microdissectionapparatus of claim 1 .
 16. An integral portion of a biological reactionvessel, comprising: a transfer film carrier having a substrate surface;and a laser capture microdissection transfer film coupled to saidsubstrate surface of said transfer film carrier.
 17. The integralportion of a biological reaction vessel according to claim 16 , whereinsaid laser capture microdissection transfer film includes a material,that upon exposure to sufficient electromagnetic energy, expands andprojects itself away from said substrate surface.
 18. The integralportion of a biological reaction vessel according to claim 16 , furthercomprising a scattering media in proximity to said laser capturemicrodissection transfer film.
 19. The integral portion of a biologicalreaction vessel according to claim 16 , wherein said laser capturemicrodissection transfer film includes an absorptive substance.
 20. Theintegral portion of a biological reaction vessel according to claim 16 ,wherein said laser capture microdissection transfer film is hot vacuumbaked onto said substrate surface.
 21. The integral portion of abiological reaction vessel according to claim 16 , wherein said lasercapture microdissection transfer film is bonded to said substratesurface with a refractive index matching transparent glue.
 22. Theintegral portion of a biological reaction vessel according to claim 16 ,wherein said transfer film carrier includes a negative draft such that adistal diameter defined by said surface of said transfer film carrier isgreater than a proximal diameter defined by an inner perimeter of saidtransfer film carrier.
 23. The integral portion of a biological reactionvessel according to claim 22 , wherein said transfer film carrierincludes a girdle that is contiguous with said negative draft.
 24. Theintegral portion of a biological reaction vessel according to claim 22 ,wherein said transfer film carrier includes a chamfer that is contiguouswith said substrate surface.
 25. The integral portion of a biologicalreaction vessel according to claim 16 , wherein said laser capturemicrodissection transfer film has a thickness that is less than 500microns.
 26. The integral portion of a biological reaction vesselaccording to claim 16 , wherein said laser capture microdissectiontransfer film has a thickness that is held to within 20%.
 27. Theintegral portion of a biological reaction vessel according to claim 16 ,wherein said laser capture microdissection transfer film has a surfaceopposite said substrate surface having a flatness that is held withinfive microns.
 28. The integral portion of a biological reaction vesselaccording to claim 16 , wherein said laser capture microdissectiontransfer film includes at least one pedestal that protrudes and definesa laser capture microdissection acquisition zone.
 29. The integralportion of a biological reaction vessel according to claim 16 , whereinsaid laser capture microdissection transfer film includes a protrudingfeature that runs along at least at least three points of a perimeter ofsaid laser capture microdissection transfer film.
 30. A microcentrifugetube cap comprising the integral portion of a biological reaction vesselaccording to claim 16 .
 31. A laser capture microdissection assemblycomprising: a plate having a top surface; and at least one laser capturemicrodissection cap coupled to said top surface of said plate, whereinsaid at least one laser capture microdissection cap includes a transferfilm carrier having a substrate surface; and a laser capturemicrodissection transfer film coupled to said substrate surface of saidtransfer film carrier.
 32. The laser capture microdissection assembly ofclaim 31 , further comprising a release layer coated on said plate, saidrelease layer being located between said plate and said laser capturemicrodissection transfer film of each of said at least one laser capturemicrodissection cap.
 33. The laser capture microdissection assembly ofclaim 32 , wherein said release layer includes at least one nonadhesivematerial selected from the group consisting of silicones andpolytetrafluoroethylenes.
 34. The laser capture microdissection assemblyof claim 33 , wherein said at least one nonadhesive material is asilicone containing surfactant agent.
 35. The laser capturemicrodissection assembly of claim 31 , wherein a plano-concave void islocated between said laser capture microdissection transfer film of saidat least one laser capture microdissection cap and said top surface ofsaid plate.
 36. The laser capture microdissection assembly of claim 31 ,wherein said laser capture microdissection transfer film includes atransparent thermoplastic.
 37. The laser capture microdissectionassembly of claim 31 , wherein said laser capture microdissectiontransfer film includes an absorptive substance.
 38. The laser capturemicrodissection assembly of claim 31 , wherein said laser capturemicrodissection transfer film is hot vacuum baked onto said substratesurface.
 39. The laser capture microdissection assembly of claim 31 ,wherein said transfer film carrier includes a negative draft such that adistal diameter defined by said surface of said transfer film carrier isgreater than a proximal diameter defined by an inner perimeter of saidtransfer film carrier.
 40. The laser capture microdissection assembly ofclaim 31 , wherein said laser capture microdissection transfer film hasa thickness that is less than 500 microns.
 41. The laser capturemicrodissection assembly of claim 31 , wherein said laser capturemicrodissection transfer film has a thickness that is held to within 20%of a given value.
 42. The laser capture microdissection assembly ofclaim 31 , wherein said laser capture microdissection transfer film hasa surface opposite said substrate surface having a flatness that is heldwithin five microns.
 43. The laser capture microdissection assembly ofclaim 31 , further comprising at least one diffuser coupled to said atleast one transfer film carrier.
 44. A set of microcentrifuge tube capscomprising the laser capture microdissection assembly of claim 31 . 45.A method of imaging a sample with a microscope, comprising: providingsaid microscope; locating a scattering media within a beam path definedby said microscope and within a few millimeters of a sample; and imagingsaid sample through said scattering media with said microscope.
 46. Themethod of imaging a sample with a microscope according to claim 45 ,wherein said scattering media is optically coupled to a laser capturemicrodissection film.
 47. A microscope, comprising: a scattering medialocated within a beam path defined by said microscope and within a fewmillimeters of a sample.
 48. The microscope of claim 47 , furthercomprising a laser capture microdissection film optically coupled tosaid scattering media.